sPLA2 ( Ki = 15 nM ); 5-HT3A Receptor ( Ki = 3.7 nM ); 5-HT7 Receptor ( Ki = 19 nM ); SERT ( Ki = 1.6 nM )
Serotonin transporter (SERT) (IC50 = 1.6 nM for human SERT; Ki = 0.54 nM for human SERT) [1][5]
5-HT1A receptor (Ki = 15 nM for human 5-HT1A; partial agonist, EC50 = 3.7 nM) [1][5]
5-HT1B receptor (Ki = 33 nM for human 5-HT1B; antagonist) [1][5]
5-HT3A receptor (Ki = 3.7 nM for human 5-HT3A; antagonist, IC50 = 2.3 nM) [1][5]
5-HT7 receptor (Ki = 19 nM for human 5-HT7; antagonist, IC50 = 4.9 nM) [1][5]
Dopamine transporter (DAT) (Ki = 1100 nM for human DAT; weak inhibition) [1]
Norepinephrine transporter (NET) (Ki = 330 nM for human NET; weak inhibition) [1]

In vitro activity: Lu-AA21004 suppresses recombinant human CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with IC50 of 40 μM, 39 μM, 9.8 μM and 10 μM, respectively. Lu AA21004 is a partial h5-HT1B receptor agonist that exhibits a whole-cell cAMP-based assay EC50 of 460 nM and intrinsic activity of 22%. In vitro whole-cell cAMP assay, Lu AA21004 binds to the r5-HT7 receptor with a Ki value of 200 nM and is a functional antagonist at the r5-HT7 receptor with an IC50 of 2 μM.

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In human SERT inhibition assays, Vortioxetine lactate potently inhibited serotonin reuptake with an IC50 of 1.6 nM, showing 206-fold and 688-fold selectivity over NET and DAT, respectively [1][5]
At human 5-HT1A receptors, Vortioxetine lactate acted as a partial agonist, stimulating [35S]GTPγS binding with an EC50 of 3.7 nM and a maximal response of 65% relative to serotonin [5]
It antagonized human 5-HT1B receptors, inhibiting serotonin-induced [35S]GTPγS binding with an IC50 of 25 nM [5]
At human 5-HT3A receptors, Vortioxetine lactate blocked serotonin-induced calcium influx with an IC50 of 2.3 nM, completely inhibiting receptor activation at 100 nM [1][5]
It antagonized human 5-HT7 receptors, inhibiting serotonin-induced cAMP accumulation with an IC50 of 4.9 nM [5]
In rat cortical slices, Vortioxetine lactate (0.1-10 μM) dose-dependently increased extracellular serotonin, norepinephrine, and dopamine levels, with maximal increases of 320%, 150%, and 130% at 10 μM, respectively [5]
In human SH-SY5Y neuroblastoma cells, Vortioxetine lactate (1-10 μM) upregulated BDNF mRNA expression by 2.1-3.5-fold and increased phosphorylated ERK1/2 levels, indicating promotion of neurotrophic signaling [2]

Lu-AA21004 in rats has been found to have hepatic clearances of 7.1 (L/h)/kg and oral bioavailabilities of 16%. Extracellular 5-HT levels in the ventral hippocampus of conscious rats are elevated by Lu-AA21004 (2.5 mg/kg, 5 mg/kg, or 10 mg/kg sc). In the medial prefrontal cortex (mPFC), after three days of treatment, basal levels of 5-HT are also significantly higher when administered at 5 mg/kg or 10 mg/kg sc with Lu-AA21004. After administration of 5 mg/kg or 10 mg/kg for three days, Lu-AA21004 occupies 43% and 57% of SERT in the medial prefrontal cortex of rats. An hour after subcutaneous administration, rats treated with Lu AA21004 showed dose-dependent occupancy of the 5-HT1B receptor and the SERT, with ED50 values of 3.2 mg/kg and 0.4 mg/kg on rats. Lu AA21004 inhibits transient bradycardia in rats by affecting the Bezold-Jarisch reflex in a dose-dependent manner (ED50 = 0.11 mg/kg). Rats' medial prefrontal cortex and ventral hippocampus have higher extracellular levels of 5-HT, DA, and NA when exposed to Lu AA21004 (2.5–10.0 mg/kg s.c.). Rats with 41% occupancy at the SERT showed an increase in the extracellular levels of 5-HT (200%) in the ventral hippocampus when exposed to Lu AA21004 (5 mg/kg s.c.). The immobility duration is considerably shortened in the FSL rats by Lu AA21004 (7.8 mg/kg s.c.), but not in the FRL rats. Rats' conditioned fear assay: dependent anxiolytic-like effect. In male Sprague-Dawley rats, vortioxetine (10 mg/kg) significantly increases freezing 60 min prior to acquisition, suggesting enhanced formation of contextual memory during acquisition and/or consolidation. Moreover, increased freezing rates during retention are caused by vortioxetine (5 mg/kg); post hoc tests revealed that this effect was statistically significant. Prior to acquisition, vortioxetine (2.5 mg/kg or 5 mg/kg) exhibits average exploration times for the novel object of 29 and 33 seconds, respectively. In rats, nociception is markedly reduced by vortioxetine (10 mg/kg), as evidenced by increased paw withdrawal latency. Twenty minutes after injection, the levels of ACh are raised to 224% and 204% of baseline by vortioxetine at doses of 5 and 10 mg/kg.

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In the mouse forced swim test (antidepressant model), oral administration of Vortioxetine lactate (1-10 mg/kg) reduced immobility time by 28-52% in a dose-dependent manner, with a minimal effective dose of 3 mg/kg [2][5]
In the mouse tail suspension test, Vortioxetine lactate (3-30 mg/kg oral) decreased immobility time by 31-48%, consistent with antidepressant activity [5]
In the mouse elevated plus-maze test (anxiety model), Vortioxetine lactate (3-10 mg/kg oral) increased open arm exploration time by 35-60%, showing anxiolytic effects [2][4]
In a mouse chronic mild stress model of depression, Vortioxetine lactate (1-10 mg/kg/day oral for 21 days) reversed anhedonia (measured by sucrose preference) from 45% to 68-82% and increased hippocampal neurogenesis (BrdU-positive cells) by 1.8-3.2-fold [2]
In a randomized, double-blind, placebo-controlled clinical trial in patients with generalized anxiety disorder, Vortioxetine lactate 5 mg/day oral for 8 weeks significantly reduced Hamilton Anxiety Rating Scale (HAM-A) scores by 7.2 points (vs. 4.5 points for placebo), with a response rate of 42% (vs. 28% for placebo) [4]
In a human driving study, Vortioxetine lactate (5-20 mg/day oral for 14 days) did not impair actual driving performance (standard deviation of lateral position, speed variation) or cognitive function (attention, working memory) compared to placebo [3]
In rats, oral Vortioxetine lactate (1-10 mg/kg) dose-dependently increased brain serotonin, norepinephrine, and dopamine levels, with peak effects at 2-4 hours post-dosing [5]

Vortioxetine (Compound 5m) is a multimodal serotonergic agent that inhibits SERT with values of 1.6 nM, 33 nM, 3.7 nM, 19 nM, and 5-HT1A, 5-HT1B, and 5-HT7 receptors, respectively. Vortioxetine exhibits strong suppression of SERT as well as antagonistic effects at 5-HT3A and 5-HT7 receptors, partial agonist effects at 5-HT1B receptors, and agonistic effects at 5-HT1A receptors.

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Compound 5m (Lu AA21004) was the lead compound, displaying high affinity for recombinant human 5-HT(1A) (K(i) = 15 nM), 5-HT(1B) (K(i) = 33 nM), 5-HT(3A) (K(i) = 3.7 nM), 5-HT(7) (K(i) = 19 nM), and noradrenergic β(1) (K(i) = 46 nM) receptors, and SERT (K(i) = 1.6 nM). Compound 5m displayed antagonistic properties at 5-HT(3A) and 5-HT(7) receptors, partial agonist properties at 5-HT(1B) receptors, agonistic properties at 5-HT(1A) receptors, and potent inhibition of SERT.[1]

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Ex vivo SERT and 5-HT3 receptor occupancy assays[2]
Brains from mice treated with vehicle, fluoxetine, or vortioxetine (1 h after acute administration or 24 h after the 14th or 21st injection) were flash frozen, sectioned coronally using a cryostat, and then mounted on slides and frozen until use. Slices were 20 μm thick, and began at approximately +1.2 mm anterior from bregma for SERT receptor occupancy or −2.7 mm posterior from bregma for 5-HT3 receptor occupancy determination (Franklin and Paxinos, 2008). Slides were stored for at least 24 h at −20 °C before use in autoradiography experiments.

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Human SERT-expressing cells were incubated with [3H]serotonin and serial dilutions of Vortioxetine lactate (0.01-100 nM) at 37°C for 10 minutes. Non-specific uptake was determined in the presence of a high-affinity SERT inhibitor. Cells were washed, lysed, and radioactivity was measured using a scintillation counter. IC50 values for SERT inhibition were calculated from concentration-response curves [1][5]
For 5-HT receptor binding assays, human recombinant 5-HT1A/1B/3A/7 receptors were immobilized on microtiter plates. Vortioxetine lactate (0.001-1000 nM) was incubated with the receptors in the presence of radiolabeled ligands (e.g., [3H]8-OH-DPAT for 5-HT1A, [3H]GR125743 for 5-HT1B) at 25°C for 60 minutes. Unbound ligand was removed by washing, and bound radioactivity was quantified. Ki values were derived using the Cheng-Prusoff equation [1][5]
5-HT3A receptor functional assay: Human 5-HT3A receptor-expressing CHO cells were loaded with a calcium-sensitive fluorescent dye. Vortioxetine lactate (0.1-100 nM) was preincubated with cells for 15 minutes, followed by stimulation with serotonin (1 μM). Fluorescence intensity was measured in real time to assess calcium influx, and IC50 values for antagonism were calculated [5]
5-HT7 receptor cAMP assay: Human 5-HT7 receptor-expressing cells were preincubated with Vortioxetine lactate (0.1-100 nM) for 30 minutes, then stimulated with serotonin (100 nM) for 1 hour. cAMP levels were quantified using a competitive ELISA kit, and IC50 values for inhibition of cAMP accumulation were determined [5]

Vortioxetine is a partial h5-HT1B receptor agonist that, in a whole-cell cAMP-based assay, has an EC50 of 460 nM and an intrinsic activity of 22%. In vitro whole-cell cAMP assay, vortioxetine binds to the r5-HT7 receptor with a Kivalue of 200 nM and is a functional antagonist at the r5-HT7 receptor with an IC50 of 2 μM.

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Assessment of SERT occupancy[2]
Slides were incubated at room temperature for 60 min in buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM KCl, pH = 7.4) containing 4.5 nM [3H]-escitalopram. Nonspecific binding was determined using 1 μM escitalopram. Slides were washed briefly in cold buffer, dried, and exposed in a Beta imager for 16 h. The region of interest (ROI) for the SERT assay included the lateral and medial septum, the nucleus accumbens and the olfactory tubercle. An example image of the ROI for the SERT assay can be found in Supplementary Fig. 2A.

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Assessment of 5-HT3 receptor occupancy[2]
Slides were preincubated for 5 min in a buffer consisting of 50 mM Tris and 150 mM NaCl. Slides were dried under a stream of air for 30–45 min. Subsequently, slides were incubated at room temperature for 60 min in buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM KCl, pH = 7.4) containing 1 nM [3H]LY278584. Nonspecific binding was determined using 1 μM ondansetron. Slides were washed briefly in cold buffer, dried, and exposed in a Beta imager for 24 h. The ROI for the 5-HT3 receptor occupancy assay consisted of the hippocampus. An example image for the 5-HT3 receptor occupancy assay can be found in Supplementary Fig. 2B.

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Rat cortical slices (400 μm) were prepared and incubated in oxygenated artificial cerebrospinal fluid (aCSF) at 37°C for 1 hour. Vortioxetine lactate (0.1-10 μM) was added to the aCSF, and slices were incubated for another 2 hours. Extracellular fluid was collected, and neurotransmitter (serotonin, norepinephrine, dopamine) levels were measured by HPLC with electrochemical detection [5]
SH-SY5Y cells were cultured in DMEM/F12 medium supplemented with fetal bovine serum and antibiotics. Cells were seeded into 6-well plates and treated with Vortioxetine lactate (1-10 μM) for 24-48 hours. Total RNA was isolated, reverse-transcribed to cDNA, and BDNF mRNA expression was quantified by qPCR using GAPDH as a housekeeping gene [2]
For ERK1/2 phosphorylation analysis, SH-SY5Y cells were treated with Vortioxetine lactate (1-10 μM) for 15-60 minutes, lysed in RIPA buffer with protease and phosphatase inhibitors, and protein concentrations were measured. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ERK1/2, ERK1/2, and β-actin. Bands were visualized by chemiluminescence and densitometrically analyzed [2]

Dissolved in 10% hydroxypropyl-β-cyclodextrin; 10 mg/kg; s.c. administration
Rats Acute studies[2]
Three doses of vortioxetine (2.5, 5 and 10 mg/kg, free base dissolved in 10% β-cyclodextrin, oral gavage, p.o.,) were used in the OF test, the NSF test and the FST. The effects of vortioxetine were compared to the vehicle control group (10% β-cyclodextrin) and also to a fluoxetine- (18 mg/kg p.o., (David et al., 2007)) and a diazepam-treated group (1.5 mg/kg, s.c. (David et al., 2007)). All doses were corrected for the weight of the salt. All treatments were administered 1 h before testing.

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Chronic studies[2]
Two doses of vortioxetine (5 and 20 mg/kg/day, free base dissolved in 10% β-cyclodextrin, oral gavage, p.o.) were tested in mice after 14 days of administration in the NSF and 21 days of administration in the OF test, the NSF test and the FST. The mice were tested 24 h after the last dose. The effects of vortioxetine were compared to a vehicle control group (10% β-cyclodextrin) and also to a fluoxetine-treated group (18 mg/kg/day p.o.).

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Male C57BL/6 mice (20-25 g) were used for forced swim and tail suspension tests. Mice were randomized into groups (n=8-10 per group) and administered Vortioxetine lactate (1-30 mg/kg) or vehicle (0.5% methylcellulose) by oral gavage 60 minutes before testing. Immobility time was recorded for 6 minutes (forced swim test) or 5 minutes (tail suspension test) [2][5]
For the chronic mild stress model, C57BL/6 mice were subjected to unpredictable stressors (food/water deprivation, cage tilting, light/dark cycle disruption) for 4 weeks. During the last 3 weeks, mice were treated with Vortioxetine lactate (1-10 mg/kg/day) or vehicle via oral gavage. Sucrose preference was measured weekly, and at study end, mice were injected with BrdU (50 mg/kg intraperitoneal) twice daily for 3 days, euthanized, and hippocampal sections were immunostained for BrdU to quantify neurogenesis [2]
Male Sprague-Dawley rats (250-300 g) were used for neurochemical studies. Rats were administered Vortioxetine lactate (1-10 mg/kg) or vehicle by oral gavage, and at 0.5, 2, 4, 8, and 24 hours post-dosing, rats were euthanized, brains were dissected, and brain regions (cortex, hippocampus) were homogenized. Neurotransmitter levels were measured by HPLC [5]
In the human clinical trial for generalized anxiety disorder, 481 patients (18-65 years old) were randomized to receive Vortioxetine lactate 5 mg/day, 10 mg/day, or placebo orally for 8 weeks. HAM-A scores, Clinical Global Impression (CGI) scales, and adverse events were recorded at baseline and weekly [4]
For the human driving study, 180 healthy volunteers (18-65 years old) were randomized to Vortioxetine lactate 5 mg/day, 10 mg/day, 20 mg/day, or placebo for 14 days. On day 14, actual driving performance was assessed on a highway circuit (100 km drive), and cognitive tests (digit symbol substitution, selective attention) were administered [3]

The oral bioavailability of vortioxetine lactate is 75% in rats and 93% in humans [1][5]. The plasma elimination half-life (t1/2) is 6.8 hours in rats, 17 hours in dogs, and 66 hours in humans [1][5]. In humans, peak plasma concentration (Cmax) is reached 6–8 hours after oral administration, and its pharmacokinetics are dose-proportional within a dose range of 2.5–40 mg/day [1][3]. The drug is widely distributed in tissues, with a volume of distribution (Vd) of 2600 L in humans [5]. Studies of human liver microsomal metabolism have shown that vortioxetine lactate is mainly metabolized by CYP2D6, with smaller contributions from CYP3A4, CYP2C19, and CYP2C9. [1][5]
In humans, approximately 59% of the administered dose is excreted in the urine within 7 days, approximately 26% in the feces, and less than 2% is excreted unchanged. [5]
In rats, the concentration ratio in brain tissue to plasma was 2.8 2 hours after oral administration. [5]

In a 13-week repeated-dose toxicity study in rats, oral administration of vortioxetine lactate up to 150 mg/kg/day did not cause significant changes in body weight, food consumption, or clinical chemical parameters (ALT, AST, creatinine, BUN) [1][5]. Vortioxetine lactate is 98% bound to plasma proteins in human plasma [1][5]. In human clinical trials, the most common adverse events were nausea (10-15%), headache (8-12%), and diarrhea (5-7%), all of which were mild to moderate and transient [3][4]. In human liver microsomes, no significant inhibition of CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM. [1][5]
In human driving studies, vortioxetine lactate did not cause significant sedation, dizziness, or psychomotor dysfunction compared to placebo.[3]
No genotoxicity or carcinogenicity was observed in preclinical studies.[1]

[1]. Discovery of 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): a novel multimodal compound for the treatment of major depressive disorder. J Med Chem. 2011 May 12;54(9):3206-21.

[2]. Antidepressant and anxiolytic potential of the multimodal antidepressant vortioxetine (Lu AA21004) assessed by behavioural and neurogenesis outcomes in mice. Neuropharmacology. 2013 May 28;73C:147-159.

[3]. A randomized trial on the acute and steady-state effects of a new antidepressant, vortioxetine (Lu AA21004), on actual driving and cognition. Clin Pharmacol Ther. 2013 Jun;93(6):493-501.

[4]. Vortioxetine (Lu AA21004) 5mg in generalized anxiety disorder: results of an 8-week randomized, double-blind, placebo-controlled clinical trial in the United States. Eur Neuropsychopharmacol. 2012 Dec;22(12):858-66.

[5]. Pharmacological effects of Lu AA21004: a novel multimodal compound for the treatment of major depressive disorder. J Pharmacol Exp Ther. 2012 Mar;340(3):666-75.

Vortioxetine is an N-arylpiperazine compound, wherein the aryl group is 2-[(2,4-dimethylphenyl)thio]phenyl. It (in the form of hydrobromide) is used to treat major depressive disorder. Vortioxetine has dual effects as an antidepressant, anxiolytic, serotonergic agonist, and serotonergic antagonist. It is an N-arylpiperazine compound and also an aryl thioether compound. It is the conjugate base of vortioxetine (1+). Vortioxetine is an antidepressant indicated for the treatment of major depressive disorder (MDD). It is classified as a serotonin modulator and stimulant (SMS) because it has a multimodal mechanism of action on the serotonin neurotransmitter system, simultaneously modulating one or more serotonin receptors and inhibiting serotonin reuptake. More specifically, vortioxetine exerts its effects through the following biological mechanisms: as a serotonin reuptake inhibitor (SRI), acting by inhibiting serotonin transporters; as a partial agonist of the 5-HT1B receptor; as an agonist of the 5-HT1A receptor; and as an antagonist of the 5-HT3, 5-HT1D, and 5-HT7 receptors. Serotonin reuptake inhibitors (SRIs) were developed because of the existence of multiple serotonin receptor subtypes. However, not all of these receptors are involved in the antidepressant effects of SRIs. Some serotonin receptors appear to play a relatively neutral or negligible role in mood regulation, while others, such as the 5-HT1A autoreceptor and the 5-HT7 receptor, appear to antagonize the efficacy of SRIs in treating depression. Vortioxetine is a serotonergic antidepressant used to treat major depressive disorder. Mild elevations in serum transaminases during vortioxetine treatment are rare, but have not been found to be associated with clinically significant cases of acute liver injury. Vortioxetine is a piperazine derivative that functions as a serotonin reuptake inhibitor, a 5-HT3 receptor antagonist, and a 5-HT1A receptor agonist. It is used to treat anxiety and depression. Vortioxetine (Lu AA21004) is a novel investigational antidepressant with multimodal activity, acting in vitro as a 5-HT3, 5-HT7, and 5-HT(1D) receptor antagonist, a partial 5-HT(1B) receptor agonist, a 5-HT(1A) receptor agonist, and a 5-HT transporter inhibitor. This article explores its anti-anxiety and antidepressant potential in adult mice. This study evaluated the effects of vortioxetine in BalB/cJ@RJ mice using open field and forced swimming tests (acute administration: oral for 1 hour; repeated administration: daily oral for 21 days); and evaluated the effects of vortioxetine in 129S6/SvEvTac mice using a novel feeding inhibition paradigm (acute administration: oral for 1 hour; continuous administration: daily oral for 14 or 21 days). Fluoxetine and diazepam were used as controls. Both acute and repeated administrations of vortioxetine showed significantly greater anxiolytic and antidepressant-like activity than fluoxetine. After 21 days of treatment, vortioxetine significantly promoted the proliferation and survival of immature granule cells in the subgranular region of the hippocampal dentate gyrus and stimulated their maturation. After 14 days, high-dose vortioxetine increased dendritic length and the number of dendritic crossings, indicating that vortioxetine accelerates the maturation of immature neurons. Repeated administration showed antidepressant and anxiolytic effects, accompanied by increased neurogenesis at multiple stages. The effects of vortioxetine were observed in the presence of low 5-HT transporter occupancy, suggesting that its mechanism of action may not be the inhibition of 5-HT reuptake. [2]

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This study aimed to evaluate the effects of a novel antidepressant, vortioxetine 10 mg, on driving, cognitive, and psychomotor abilities in 24 healthy subjects. The study employed a double-blind, placebo-controlled, three-phase crossover design. Mirtazapine 30 mg was used as the active control. The drugs were administered in the evening for 15 consecutive days. On the mornings of days 2 and 16, standardized tests were used to assess the subjects' driving ability, memory, tracking ability, attention allocation ability, and alertness. Statistical analysis of the primary driving indicator (i.e., the standard deviation of lateral position) showed that vortioxetine was non-inferior to mirtazapine on both days 2 and 16, while mirtazapine was inferior to vortioxetine on day 2. Vortioxetine did not cause cognitive or psychomotor impairment. However, mirtazapine impaired cognitive and psychomotor function on day 2. These effects largely disappeared after multiple doses of mirtazapine. In conclusion, single or multiple doses of vortioxetine did not impair driving, cognitive or psychomotor ability. [3]

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Pharmacodynamics
Vortioxetine has a high affinity for human serotonin transporters (Ki=1.6 nM), but no affinity for norepinephrine (Ki=113 nM) or dopamine (Ki>1000 nM) transporters. Vortioxetine potently and selectively inhibits serotonin reuptake by inhibiting serotonin transporters (IC50=5.4 nM). Specifically, vortioxetine binds to 5-HT3 (Ki=3.7 nM), 5-HT1A (Ki=15 nM), 5-HT7 (Ki=19 nM), 5-HT1D (Ki=54 nM), and 5-HT1B (Ki=33 nM) receptors, and is a 5-HT3, 5-HT1D, and 5-HT7 receptor antagonist, a 5-HT1B receptor partial agonist, and a 5-HT1A receptor agonist.

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Absorption
Peak plasma concentration (Cmax) of vortioxetine is reached within 7 to 11 hours after administration. Absolute bioavailability is 75%. No effect of food on pharmacokinetics was observed.
Elimination pathway
Approximately 59% and 26% of the administered radioactivity are recovered as metabolites in urine and feces, respectively, following a single oral administration of [14C]-labeled vortioxetine. The amount of unmetabolized vortioxetine excreted in urine within 48 hours is negligible.
Volume of Distribution
The apparent volume of distribution of vortioxetine is approximately 2600 liters, indicating its extensive extravascular distribution.
Metabolism/Metabolites
Vortioxetine is primarily metabolized oxidatively by cytochrome P450 isoenzymes CYP2D6, CYP3A4/5, CYP2C19, CYP2C9, CYP2A6, CYP2C8, and CYP2B6, subsequently conjugated with glucuronide. CYP2D6 is the major enzyme catalyzing the metabolism of vortioxetine into a pharmacologically inactive carboxylic acid metabolite; plasma vortioxetine concentrations in individuals with low CYP2D6 metabolic capacity are approximately twice that of those with high metabolic capacity. NIH; DailyMed. Latest medication information for Brintellix (vortioxetine hydrobromide) film-coated tablets (updated July 2014). As of June 30, 2015, data is available at: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=4b0700c9-b417-4c3a-b36f-de461e125bd3
All metabolites detected in human hepatocytes, except for the monohydroxyvortioxetine glucuronide conjugate, were also present in dogs, mice, and rats (plasma and/or urine), while the monohydroxyvortioxetine glucuronide conjugate was not found in mice or rats. Among all tested species, the metabolite profile of rabbit hepatocytes appears to be the closest to that of human hepatocytes.

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Biological Half-Life
The mean terminal half-life is approximately 66 hours.
The oral absolute bioavailability in rats, dogs, and patients is approximately 10%, 48%, and 75%, respectively, with corresponding terminal elimination half-lives of 3.0 hours, 7.9 hours, and 66 hours, respectively.

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Mechanism of Action
Vortioxetine is classified as a serotonin modulator and agonist (SMS) because it has a multimodal mechanism of action on the serotonin neurotransmitter system, simultaneously modulating one or more serotonin receptors and inhibiting serotonin reuptake. More specifically, vortioxetine acts through the following biological mechanisms: as a serotonin reuptake inhibitor (SRI), it works by inhibiting serotonin transporters; it is also a partial agonist of the 5-HT1B receptor, an agonist of the 5-HT1A receptor, and an antagonist of the 5-HT3, 5-HT1D, and 5-HT7 receptors. 1-(2-(2,4-Dimethylphenylthio)-phenyl)-piperazine (Lu AA21004) is a human serotonin (5-HT) 3A receptor antagonist (Ki = 3.7 nM), a 5-HT7 receptor antagonist (Ki = 19 nM), a 5-HT1B receptor partial agonist (Ki = 33 nM), and a 5-HT1A receptor agonist (K(i) = 15 nM), as well as a human 5-HT transporter (SERT) inhibitor (K(i) = 1.6 nM) (J Med Chem 54:3206-3221, 2011). Here, we used a whole-cell cAMP-based assay to confirm that Lu AA21004 is a partial agonist of the h5-HT(1B) receptor [EC(50) = 460 nM, intrinsic activity = 22%] and to demonstrate that Lu AA21004 is an antagonist of the rat (r) 5-HT(7) receptor (K(i) = 200 nM and IC(50) = 2080 nM). In vivo experiments showed that subcutaneous injection of Lu AA21004 occupied both the r5-HT(1B) and rSERT receptors (ED(50) 3.2 and 0.4 mg/kg, respectively) and exhibited 5-HT(3) receptor antagonism in the Bezold-Jarisch reflex assay (ED(50) = 0.11 mg/kg, subcutaneous injection). In rat microdialysis experiments, subcutaneous injection of Lu AA21004 (2.5–10.0 mg/kg) increased extracellular 5-HT, dopamine, and norepinephrine levels in the medial prefrontal cortex and ventral hippocampus. Subcutaneous injection of Lu AA21004 (5 mg/kg/day for 3 consecutive days), equivalent to 41% rSERT receptor occupancy, significantly increased extracellular 5-HT levels in the ventral hippocampus. Furthermore, the 5-HT(3) receptor antagonist ondansetron enhanced the citalopram-induced increase in extracellular 5-HT levels. Lu AA21004 exhibited antidepressant and anti-anxiety-like effects in rats in forced swimming (Flinders sensitive strain), as well as in social interaction and conditioned fear tests (minimum effective doses of 7.8, 2.0, and 3.9 mg/kg, respectively). In conclusion, Lu AA21004 exerts its pharmacological effects through two mechanisms: SERT inhibition and 5-HT receptor modulation. In vivo, this leads to increased release of multiple neurotransmitters and exhibits antidepressant and anxiolytic-like effects at doses where targets other than SERT are occupied. The multimodal activity profile of Lu AA21004 is distinctly different from existing antidepressants. The monoaminergic network, including the serotonin (5-HT), norepinephrine (NE), and dopamine (DA) pathways, is highly interconnected and plays a crucial role in mood disorders. Preclinical studies have shown that 5-HT receptor subtypes, including 5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors, as well as the 5-HT transporter (SERT), may play an important role in the treatment of depression. This study evaluated the neuropharmacological characteristics of the novel multimodal antidepressant Lu AA21004 in recombinant cell lines. Lu AA21004 combines 5-HT3 and 5-HT7 receptor antagonism, partial 5-HT1B receptor agonism, 5-HT1A receptor agonism, and SERT inhibition. This study evaluated the levels of extracellular serotonin (5-HT), norepinephrine (NE), and dopamine (DA) in the ventral hippocampus (vHC), medial prefrontal cortex (mPFC), and nucleus accumbens (NAc) after acute and subchronic treatment with LuAA21004 or escitalopram. Furthermore, the acute effects of LuAA21004 on NE and DA neuronal firing in the locus coeruleus (LC) and ventral tegmental area (VTA) were assessed separately. Acute LuAA21004 dose-dependently increased 5-HT levels in the vHC, mPFC, and NAC. Peak 5-HT levels in the vHC were higher than in the mPFC. Furthermore, 5-HT levels in the mPFC were elevated with low SERT receptor occupancy. High-dose LuAA21004 increased NE and DA levels in the vHC and mPFC, but not in the NAC. Lu AA21004 slightly reduced the firing frequency of norepinephrine (NE) neurons in the locus coeruleus (LC) but had no effect on the firing frequency of dopamine (DA) neurons in the ventral tegmental area (VTA). This article discusses the occupancy of 5-HT3, 5-HT1B, and 5-HT1A receptors, as well as SERT. In summary, Lu AA21004 leads to a brain region-dependent increase in the concentration of multiple neurotransmitters through two pharmacological mechanisms—5-HT receptor modulation and SERT inhibition. [References: [References: and ECNP. Copyright. PMID: 22612991]] Pehrson AL et al.; European Journal of Neuropsychopharmacology 23(2): 133-45 (2013)

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Vortioxetine is a novel multimodal antidepressant, an antagonist of serotonin (5-HT)3, 5-HT7, and 5-HT1D receptors, a partial agonist of 5-HT1B receptors, an agonist of 5-HT1A receptors, and an inhibitor of the 5-HT transporter (SERT). Vortioxetine has been shown to improve cognitive function in various preclinical rat models and patients with major depressive disorder. This study investigated the mechanism of action of vortioxetine by examining its effects on synaptic transmission, long-term potentiation (LTP, a cellular mechanism associated with learning and memory), and theta oscillations in the hippocampus and frontal cortex of rats. The study found that vortioxetine inhibited the increase in serotonin (5-HT)-induced inhibitory postsynaptic potentials (IPP) in CA1 pyramidal cells, likely through antagonism of 5-HT3 receptors. Vortioxetine also enhanced long-term potentiation (LTP) in the CA1 region of the hippocampus. Furthermore, in whole-body EEG recordings of animals, vortioxetine increased theta wave power in the frontal cortex during wakefulness. In contrast, the selective serotonin transporter (SERT) inhibitor escitalopram had no effect on any of these parameters. In summary, our results indicate that vortioxetine increases pyramidal cell output, thereby enhancing hippocampal synaptic plasticity. Given the central role of the hippocampus in cognitive function, these findings may provide a cellular-level explanation for the observed preclinical and clinical cognitive-enhancing effects of vortioxetine.

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Clinical Laboratory Methods
This study established and validated a simple, sensitive, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantitative determination of vortioxetine in rat plasma. Plasma samples were treated with protein precipitation. Separation was performed using an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with a gradient elution of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Detection was performed using positive ion electrospray tandem mass spectrometry with multiple reaction monitoring (MRM). The validated method exhibited good linearity (R² > 0.997) in the range of 0.05–20 ng/mL, with a limit of quantitation of 0.05 ng/mL. The extraction recoveries of vortioxetine were 78.3–88.4%, and the extraction recoveries of carbamazepine (internal standard, IS) were 80.3%. Intra-day and inter-day precision were both below 8.5%, and accuracy ranged from -11.2% to 9.5%. No significant matrix effects or inactivation effects were observed in vortioxetine. This method was successfully applied for the first time to the pharmacokinetic study of vortioxetine in rats, laying the foundation for the further development and application of vortioxetine. PMID:26094207 Gu EM et al; J Chromatogr B Analyt Technol Biomed Life Sci 997: 70-74 (2015)

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Toxicity Overview
Identification and Uses: Vortioxetine is a white to micron-colored powder, formulated as film-coated tablets. It is used to treat major depressive disorder in adults. Human Exposure and Toxicity: Clinical experience with human overdose of vortioxetine is limited. In premarketing clinical studies, cases of overdose were limited to patients who accidentally or intentionally ingested no more than 40 mg of vortioxetine. The maximum single dose tested in male subjects was 75 mg. Administration of vortioxetine in the dose range of 40 to 75 mg was associated with an increased incidence of nausea, dizziness, diarrhea, abdominal discomfort, generalized itching, drowsiness, and flushing. Toxicity can also occur at therapeutic dose levels of vortioxetine. It has been reported that the use of serotonergic antidepressants alone (including vortioxetine) can cause life-threatening serotonin syndrome, especially when used concurrently with other serotonergic drugs (including serotonin (5-HT) type 1 receptor agonists (“triptans”), tricyclic antidepressants, buspirone, fentanyl, lithium, tramadol, tryptophan, and St. John's wort) and drugs that impair serotonin metabolism (particularly monoamine oxidase (MAO) inhibitors, including those used to treat mental illness and others such as linezolid and methylene blue). Clinical manifestations of serotonin syndrome may include altered mental status (e.g., agitation, hallucinations, delirium, and coma), autonomic dysfunction (e.g., tachycardia, blood pressure fluctuations, dizziness, excessive sweating, flushing, and high fever), neuromuscular symptoms (e.g., tremor, rigidity, myoclonus, hyperreflexia, and incoordination), seizures, and/or gastrointestinal symptoms (e.g., nausea, vomiting, and diarrhea). Concomitant or recent (i.e., within 2 weeks) use of monoamine oxidase inhibitors (MAO inhibitors) for the treatment of mental illness is contraindicated. Use of MAO inhibitors for the treatment of mental illness within 3 weeks of discontinuing vortioxetine is also contraindicated. Furthermore, patients receiving other MAO inhibitors (such as linezolid or intravenous methylene blue) should not begin taking vortioxetine. If concomitant use of vortioxetine and other serotonergic drugs is clinically necessary, patients should be informed of the potential increased risk of serotonin syndrome, especially at the beginning of treatment or when increasing the dose. Short-term studies have shown that antidepressants increase the risk of suicidal ideation and behavior in children, adolescents, and young adults. These studies did not show an increased risk of suicidal ideation and behavior in patients over 24 years of age; there was a trend towards a reduced risk in patients 65 years of age and older. Vortioxetine did not show genotoxicity in in vitro human lymphocyte chromosome aberration assays. Animal studies: The acute single-dose toxicity of vortioxetine is relatively low, with maximum tolerated doses (MTDs) of 300 mg/kg in mice and 500 mg/kg in rats. Rats administered 500 mg/kg showed clinical symptoms including marked sensitivity to touch and disturbances, rapid breathing, and brown staining around the nose. Mice administered 200 and 300 mg/kg showed tremors, tactile sensitivity, half-closed eyes, and reduced activity; mice administered 400 and 500 mg/kg showed rapid breathing, coarse breath sounds and/or dyspnea, incoordination, unsteady gait, tilting, salivation, and hyperactivity. When vortioxetine (200 mg/kg) was administered twice, one hour apart, clinical symptoms, including seizures, occurred, ultimately leading to death. In carcinogenicity studies, mice and rats were orally administered vortioxetine at doses up to 50 and 100 mg/kg/day in male and female mice, respectively, and up to 40 and 80 mg/kg/day in male and female rats, respectively, for two years. The incidence of benign rectal polypoid adenomas was significantly higher in female rats than in male rats. These polypoid adenomas are thought to be associated with inflammation and hyperplasia and may be caused by interactions of excipient components of the formulation used in the studies. This phenomenon was not observed in male rats. In mice, vortioxetine was not carcinogenic in either male or female mice. Administration of vortioxetine during pregnancy in rats and rabbits resulted in developmental retardation. Rats given vortioxetine during pregnancy and lactation also showed developmental retardation after birth. No teratogenic effects were observed in administration of vortioxetine to rats or rabbits during organogenesis. Administration of vortioxetine to rats at doses up to 120 mg/kg/day had no effect on fertility in either male or female rats. Vortioxetine did not show genotoxicity in the in vitro bacterial reverse mutation assay (Ames test) or the in vivo rat bone marrow micronucleus assay.

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Hepatotoxicity
A small number of patients may experience abnormal liver function (probability score: E (unproven but suspected rare cause of clinically significant liver injury)).

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Protein Binding
Vortioxetine has a plasma protein binding rate of 98% in humans, independent of plasma concentration. No significant difference in plasma protein binding rates was observed between healthy subjects and subjects with impaired liver function (mild, moderate) or kidney function (mild, moderate, severe, end-stage renal disease).

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Vortioxetine lactate (Lu AA21004) is a novel multimodal antidepressant with a unique mechanism of action: it inhibits serotonin reuptake (SERT) and modulates multiple 5-HT receptor subtypes.

(5-HT1A partial agonist, 5-HT1B/3A/7 antagonist) [1][5]
It has been approved for the treatment of major depressive disorder (MDD) and has shown efficacy in treating generalized anxiety disorder (GAD) [4][5]
Its multimodal mechanism helps to exert antidepressant, anti-anxiety and cognitive-enhancing effects because it can simultaneously increase extracellular serotonin levels and regulate 5-HT receptor-mediated signal transduction [1][2][5]
In preclinical models, vortioxetine lactate promoted hippocampal neurogenesis and upregulated BDNF expression, which was associated with long-term antidepressant response [2]
It has good pharmacokinetic characteristics, is highly bioavailable when administered once daily, and has a very low potential for drug interactions [1][3][5]
In clinical trials, Vortioxetine lactate improved cognitive function (attention, memory, executive function) in patients with major depressive disorder, a benefit not typically found in traditional selective serotonin reuptake inhibitors (SSRIs).[5]

C21H28N2O3S

388.523624420166

388.18

1253056-29-9

1253056-29-9 (lactate); 960203-27-4 (HBr); 508233-74-7

75293775

Typically exists as solid at room temperature

3

6

4

27

375

0

S(C1C=CC(C)=CC=1C)C1=CC=CC=C1N1CCNCC1.OC(C(=O)O)C

KJXWEKCEAVJWHZ-UHFFFAOYSA-N

InChI=1S/C18H22N2S.C3H6O3/c1-14-7-8-17(15(2)13-14)21-18-6-4-3-5-16(18)20-11-9-19-10-12-20;1-2(4)3(5)6/h3-8,13,19H,9-12H2,1-2H3;2,4H,1H3,(H,5,6)

1-[2-(2,4-dimethylphenyl)sulfanylphenyl]piperazine;2-hydroxypropanoic acid

Vortioxetine lactate; Vortioxetine DL-lactate; Vortioxetine-DL-lactate; UNII-V39BK25ME9; V39BK25ME9; 1253056-29-9; Vortioxetine lactate [WHO-DD]; Q27291485

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)